Báo cáo y học: "Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry" pdf

RESEA R C H Open Access
Biophysical analysis of HTLV-1 particles reveals
novel insights into particle morphology and
Gag stoichiometry
Iwen F Grigsby
1,2
, Wei Zhang
1,2
, Jolene L Johnson
1,4
, Keir H Fogarty
1,4
, Yan Chen
1,4
, Jonathan M Rawson
1
,
Aaron J Crosby
1
, Joachim D Mueller
1,4*
, Louis M Mansky
1,2,3*
Abstract
Background: Human T-lymphotropic virus type 1 (HTLV-1) is an important human retrovirus that is a cause of
adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle,
including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in
tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an EYFP reporter as a model
system to quantitatively analyze HTLV-1 particles released from producer cells.
Results: The codon-optimized Gag led to a dramatic and highly robust level of Gag expression as well as virus-like
particle (VLP) production. The robust level of particle production overcomes previous technical difficulties with

authentic particles and allowed for detailed analysi s of particle architecture using two novel methodologies. We
quantitatively measured the diameter and morphology of HTLV-1 VLPs in their native, hydrated state using cryo-
transmission electron microscopy (cryo-TEM). Furthermore, we were able to determine HTLV-1 Gag stoichiometry
as well as particle size with the novel biophysical technique of fluorescence fluctuation spectroscopy (FFS). The
average HTLV-1 particle diameter determined by cryo-TEM and FFS was 71 ± 20 nm and 75 ± 4 nm, respectively.
These values are significantly smaller than previou s estimates made of HTLV -1 particles by negative staining TEM.
Furthermore, cryo-TEM reveals that the majority of HTLV-1 VLPs lacks an ordered structure of the Gag lattice,
suggesting that the HTLV-1 Gag shell is very likely to be organized differently compared to that observed with HIV-
1 Gag in immature particles. This conclusion is supported by our observation that the average copy number of
HTLV-1 Gag per particle is estimated to be 510 based on FFS, which is significantly lower than that found for HIV-1
immature virions.
Conclusions: In summary, our studies represent the first quan titative biophysical analysis of HTLV-1-like particles
and reveal novel insights into particle morphology and Gag stochiometry.
Introduction
There are approximately 15-20 million people infected
by human T-lymphotropic virus type 1 (HTLV-1)
worldwide [1]. HTLV-1 infection can result in a number
of severe disorders including adult T cell leukemia/lym-
phoma (ATLL) as well as HTLV-1 associated myelopa-
thy/tropical paraparesis (HAM/TSP) [2,3]. Despite its
association with cancer and its significant impact on
human health, many of the details regarding the
replication, assembly and fundamental virus particle
structure remain poorly understood.
The Gag polyprotein is the main retroviral structural
protein and is s ufficient, in the absence of ot her viral
proteins, for the production and release of immature
VLPs [4]. The Gag polyprotein is composed of three
functional domains: mat rix (MA), caspid (CA), and
nucleocapsid (NC). Typically, upon budding or immedi-

ately after immature particle release, proteolytic cleavage
of the Gag polyproteins takes place and results in virus
particle core maturation. The Gag polyprotein is cleaved
into MA, CA, and NC by the viral protease. The newly
processed proteins reorganize into structurally distinct
* Correspondence: ;
1
Institute for Molecular Virology, University of Minnesota, Minneapolis, MN
55455, USA
Full list of author information is available at the end of the article
Grigsby et al. Retrovirology 2010, 7:75
/>© 2010 Grigsby et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommo ns.org/li censes/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provide d the original work is properly cited.
mature virions: MA remains associated with the viral
membrane; CA undergoes conformational changes and
reassembles into a viral core, which encapsulates a com-
plex of NC, genomic RNA, and other important viral
proteins [5-7].
Studies with many retroviruses, including human
immunodeficiency virus type 1 (HIV-1), have shown
that retroviral assembly is initiated by binding the myris-
toyl moiety of MA with lipid rafts at the plasma mem-
brane [8-11]. The MA-membr ane interaction is thought
to stimulate Gag oligomerization, the interaction
between viral genomic RNA and NC, a nd the recruit-
ment of a variety of host factors. Accumulation of Gag
at the plasma membrane triggers the activation of the
ESCRT machinery which creates the m embrane curva-
ture that results in the budding of immature virus parti-

cles [12]. Analysis of Gag molecules in immature HIV-1
particles have revealed that the MA domain is located at
the membrane with the CA and NC domains projecting
towards the center of the particle [13].
Cryo-electron tomography (cryo-ET) combined with
three-dimensional (3D) reconstructions have provided
highly detailed structural information for HIV-1. Struc-
tural studies have revealed that HIV-1 Gag protein s
form an incomplete paracrystalline l attice in immature
particles [14,15]. This incomplete Gag lattice was
observed to consist of a hexameric organization with
80-Å distance between neighboring ring-like structures
[14,15]. While the myristoyl moiety of MA appeared to
be associated with membrane, the hexameric ring
structure in the 3 D maps were attributed to CA, and
the Gag-Gag interactions in the immature particles
were proposed to be primarily stabilized by CA and
SP1, rather than the affinity of membrane-binding via
MA [15].
Despite limited amino acid sequence homology among
different retroviruses, the atomic tertiary structures of
individual Gag domains exhibit high similarity [16-18].
Therefore, structural and assembly mechanisms of HIV-
1 are generally used as a refere nce model for other r et-
roviruses. However, structural evidence indicates that
the conservation of Gag organization between HTLV-1
and HIV-1 is poorly understood. In this study, we have
performed cryo-TEM on HTLV-1-like particles. Our
study is the first to study HTLV-1 particles in their
nat ive, hydrated state. Our results demonstrate an aver-

age HTLV-1 particle diameter of ~ 73 nm, which is
smaller than previously predicted based on conventional
negative staining TEM [19]. Using the novel biophysical
technology of FFS, we further demonstrate that there
are ~ 510 copies of Gag per HTLV-1 particle, a number
that is significantly lower than what is typically found in
HIV-1 particles. Finally, our cryo-TEM images analysis
reveals a less ordered Gag structure compared t o that
reported for HIV-1, suggesting that the HTLV-1 Gag
shell has a distinct architecture.
Results
Creation of a tractable and robust system for the
production of HTLV-1-like particles
Previous molecular analyses of HTLV-1 replication
have been severely hampered by the fragility of HTLV-
1 proviral sequences as well as the low levels of viral
replication in tissue culture. Given the technical and
experimental limitations of working with HTLV-1, we
first sought to create an experimental model system
that would be amenable to successfully and efficiently
analyze HTLV-1 Gag trafficking and virus particle
assembly and release. It is well-established that retro-
viral Gag polyprotein is sufficient for the assembly and
release of VLPs [reviewed by [20]]. Our previous stu-
dies indicated that HTLV-1 Gag constructs express
Gag at low levels (Huating Wang and Louis Mansky,
unpublished observations), presumably due to missing
cis-elements on the RNA transcript required for effi-
cient nuclear export.
In order to c reate a tractable and robust system for

Gag expression and virus-like particle production, we
designed and created a codon-o ptimized HTLV-1 Gag
construct to improve HTLV-1 Gag expression. In order
to readily detect Gag expression, trafficking, and incor-
poration into VLPs, we fused the E YFP to the C-term-
inal end of the Gag protein. Figure 1A shows the
HTLV-1 Gag-EYFP expression construct. In this con-
struct, the Gag-EYFP isexpressedfromaCMVpromo-
ter, and a Kozak consensus sequence was engineered
upstream of the start codon to facilitate translation
initiation as well as an in-frame insertion of the EYFP
gene sequence just prior to the HTLV-1 Gag gene stop
codon. The plasmid is quite stable and readily amplified
in E. coli (data not shown).
To confirm expression of the fusion construct, 293T
cells were transiently transfected with three independent
clones of pEYFP-N3 HTLV-1 Gag in parallel experi-
ments. Thirty-six hours post-transfection, HTLV-1 Gag-
EYFP protein expression was e xamined from both cell
culture supernatants (Figure 1B, lane 1-3) and from cel-
lular lysates (Figure 1B, lane 4-6). The Gag precursor-
EYFP fusion protein, with a molecular mass of approxi-
mately 80 kDa was very readily observed, with each of
the 3 clones analyzed expressing very high and compar-
able levels of HTLV-1 Gag-EYFP. The minor bands of
smaller molecular mass likely represent partially
degraded HTLV-1 Gag-EYFP and not cleavage products
of the viral protease, since it is not pre sent in the Gag
expression construct. The Gag-EYFP observed in VLPs
was primarily full length (Figure 1B, lane 1-3), with

undetectable levels of mature capsid (p24) protein.
Grigsby et al. Retrovirology 2010, 7:75
/>Page 2 of 13
Figure 1 Development of a model system for the efficie nt expr ession of HTLV-1 Gag and robust production of VLPs. (A). HTLV-1 Gag
expression construct. The HTLV-1 Gag gene was codon-optimized with the insertion of a Kozak consensus sequence (arrow) upstream of the
ATG start codon (arrowhead). The EYFP gene was inserted in-frame prior to the Gag gene stop codon. The CMV promoter and 3’-end poly A are
indicated. (B). Immunoblot analysis of HTLV-1 Gag. An anti-HTLV-1 p24 monoclonal was used to detect HTLV-1 Gag-EYFP (arrow). Cell culture
supernatants were collected from MT-2 cells was used as a positive control. Lane 1-3 are cell culture supernatants from three independent
experiments in which pEYFP-N3-HTLV-1 Gag was transiently transfected into 293T cells; lane 4-6 are the cellular lysates. Lane “M”, molecular
markers. (C). Transmission electron microscopy of VLPs. Left panel, VLPs produced from 293T cells transiently transfected with pEYFP-N3-HTLV-1
Gag; right panel are HTLV-1 particles from MT-2 cells. Scale bar = 200 nm.
Grigsby et al. Retrovirology 2010, 7:75
/>Page 3 of 13
To investigate the morphology of the particles pro-
duced from cells expressing pEYFP-N3 HTLV-1 Gag,
transiently transfected 293T cells were harvested and
examined by TEM. MT-2 cells, a T-cell line chronically
infected by HTLV-1, were examined as a control. As
shown in Figure 1C, VLPs can be observed from 293T
cells transiently transfected with the pEYFP-N3 HTLV-1
Gag construct (Figure 1C , left panel). In comparison to
HTLV-1 produced from MT-2 cells (Figure 1C, right
panel), the VLPs produced from the fusion construct
resemble immature particles. In particular, the intense
electron density along the lipid bilayer of VLPs likely
represents the accumulation of Gag-EYFP (Figure 1C,
left inset ) in contrast to the mature viral cores observed
with HTLV-1 particl es from MT-2 cells (Figure 1C,
right inset).
We also examined the cellular localization of the Gag-

EYFP compared to Gag produced from a HTLV-1 mole-
cular clo ne. The pEYFP-N3 HTLV-1 Gag construct was
transiently transfected into HeLa cells, and 36 hours
post transfection, cells were fixed and analyzed by con-
focal microscopy (Figure 2A, B). Comparable punctate
localizat ion of Gag was observed for both the Gag-EYFP
and the Gag expressing from the full-length molecular
clone. Our observations suggest that Gag-EYFP expres-
sion in cells results in an intracellular localization pat-
tern like that of Gag produced from a HTLV-1
molecular clone. In total, our findings provide evidence
this construct results in the robust expression of HTLV-
1 Gag as well as the highly efficient production of
HTLV-1-like particles.
Analysis of HTLV-1-like particle morphology by cryo-TEM
To further characterize the VLPs produced from the
HTLV-1 Gag-EYFP expression construct, we examined
the VLP morphology by cryo-TEM. Supernatants from
293T cells transiently-cotransfected with the HTLV-1
Gag-EYFP expression construct and a VSV-G construct
were harvested, concentrated, and then subjected to a
10-40% linear sucrose gradient. The resulting VLPs were
then used in cryo-TEM. As shown in Figure 3A, the
majority of the resulting VLPs were found to be spheri-
cal, with less than 20% of the population showing an
elongated mor phology. Another example of the particles
we observed in our study is s hown in Additional file 1.
Interestingly, VLPs produced in the absence of an envel-
ope protein resulted in VLPs with irregular shapes, sug-
gesting that the envelope protein helped to stabilize the

VLP membrane (data not shown). We used the cryo-
TEM images to next measure the diameter of the VLPs,
where the average diameter was based on two measure-
ments (as illustrated in Figure 3B), with a total of 1734
particles examined. Similar to other retroviruses, there
was a range of particle size. For completeness, we
counted all particles that were spherical in shape that
appeared to have an electron dense inte rior. Using these
criteria, a total of 1734 particles were examined, ranging
from 30 to 237 nm. While the overall range of particles
observed was quite wide, the smallest (i.e., under
40 nm) and largest (i.e., over 170 nm) particles repre-
sented less than 1% of the total number of particles
observed, and their inclusion had lit tle impact on the
mean particle size (i.e., 71 +/- 20 nm versus 72 nm +/-
18). We observed that over 25% of the total population
was in the 70-80 nm range, with a mean particle size of
71 +/- 20 nm.
Analysis of VLP radial profile
We next used the informat ion obtained by cryo-TEM to
examine the VLP radial profile. For the majority of
VLPs, cryo-TEM revealed t hat the in ner Gag structure
was i ndistinguishable (Figure 3A). The partially ordered
Gag lattice can be observed (data not shown), although
the structure is less obvious compared to that reported
for HIV-1 immature particles [13]. Furthermore, the
inner density appears to vary among VLPs, with some
exhibi ting homogenous inner density, while others seem
to have an unev en distribution of electron de nsities
attributable to Gag (Figure 3A arrow).

To further analyze the electron density of VLPs, we
investigated the radial density profile of VLPs. First, the
average radial density profile was determined for se veral
particles whose diameters ranged between 70-80 nm. As
shown in Figure 4, the average distance between the
hig hest density peaks of i nner and outer leaflets of viral
membrane with MA domain is approximately 30-Å. The
MA domain is indistinguishable from the inner layer of
membrane. The electron density profile approaching the
center of the particle is relatively flat, suggesting a
homogenized inner density. Our observations indicate
that the HTLV-1-like particles are quite distinct from
those produced from HIV-1 Gag.
FFS measurement of VLP size and Gag copy number
FFS provides information about the size of a p article
through the autocorrelation function and the brightness
and concentration of the particles through the photon
counting histogram (PCH). Recent advances have
expanded this technique to allow for the examin ation of
protein oligomerization of larger complexes, including
our recent analysis of HIV-1 particles [21]. In the cur-
rent experiments, we performed measurements on the
same cell culture supernatant from 293T cells transi-
ently transfected with HTLV-1 Gag-EYFP and VSV-G
expression constructs. The supernatant from these cells
was clarified by a low-speed centrifugation to eliminate
large cell debris, and then directly used for FFS analysis.
Figure 5A shows a representative fluorescence intensity
Grigsby et al. Retrovirology 2010, 7:75
/>Page 4 of 13

A
B
Figure 2 Cellular localization of HTLV-1 Gag-EYFP and HTLV-1 Gag. HeLa cells were transiently transfected with pEYFP-N3-HTLV-1 Gag (A) or
a HTLV-1 molecular clone (B). The locations of nuclei were identified by DAPI staining (blue), HTLV-1 Gag (green). Scale bars = 28 μm.
Grigsby et al. Retrovirology 2010, 7:75
/>Page 5 of 13
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
0
10
20
30
VLP diameter (nm)
Frequency
n=1734
Mean= 71 ± 20 nm

A
(%)
B
Figure 3 Cryo-TEM analysis of HTLV-1 Gag-based VLPs. (A). Cryo-TEM images of VLPs produced from 293T cells. Examples of VLPs that have
partially occupied inner electron density are indicated with arrows. The inset shows a magnified view of a representative VLP. Scale bars = 100
nm. (B). Distribution of VLP diameter. Particle diameter was determined by averaging the longest and shortest measurements as indicated in the
diagram at the top right corner using the ImageJ software. A total of 1734 VLPs were examined (mean = 71 +/- 20 nm).
Grigsby et al. Retrovirology 2010, 7:75
/>Page 6 of 13
trace of a FFS experim ent performed on the cell culture
supernatant. The discrete fluorescence intensity spikes
are produced by VLPs passing through the observation
volume. This raw data was analyzed by fluorescence cor-
relation spectroscopy to determine the average particle
size from the autocorrelation function (Figure 5B). A fit
to a single species diffusion model accurately de scribes
the correlation function and identifies a diffusion time
of 5.2 ms. This diffusion time corresponds to an average
hydrodynamic diameter of 74 nm as determined by the
Stokes Einstein relation. Repeating the measurement
(n = 5) on independently prepared samples resulted in a
mean diameter of 75 ± 4 nm for the VLPs.
The same raw data was analyzed wit h PCH analysis to
determine the average copy number and concentration
of VLP samples. A model assuming a single VLP bright-
ness species leads to poor fits of the experimental PCH
data (reduced c
2
≥ 10). Including a s econ d VLP br ight-
ness species into the fit model was required to repro-

duce the experimental data. A fit of the photon
counting histogram to a 2-species model (reduc ed c
2
=
1.5)isshowninFigure5B.Thepresenceoftwobright-
ness species indicates brightness heterogeneity in the
VLP sample. In other words, the VLP particles passing
through the laser excitation volume are not all of equal
brightness, which gives rise to the additional br ightness
species. Each species i is characterized by its normalized
brightness b
i
and average particle number N
i
in the
observation volume. Note that the normalized bright-
ness is th e same as the Gag copy number of the VLP. It
is illustrative to briefly ignore the brightness heterogene-
itybycalculatingtheaverageGagcopynumberb
avg
of
the VLP sample according to [22].
Based on measurements of several HTLV -1-like parti-
cle samples (n = 5) we determined an average Gag copy
number per VLP of 510 ± 50 (Figure 6). To put this
number into persp ective, recall that a copy number of
~5000 Gag is required to completely fill the surface of a
140 nm HIV-1 VLP [5]. Thus, a maximum Gag copy
number of ~1300 is e xpected for the smaller (~73 nm)
HTLV-1 VLP assuming that both Gag proteins occupy a

comparable surface area at the membrane. The observa-
tion of an average Gag copy number of 510 indicates
that, on average, Gag at the membrane only covers
about half of the available surface area.
TheaverageGagcopynumberwasdeterminedfrom
the two brightness species identified by PCH analysis.
Repeated measurements of multiple independent sample
preparations confirmed the presence of the two species.
Their brightness values, which typically varied very little
across experiments, correspond to Gag copy num bers of
b
1
=300±60andb
2
= 880 ± 100 (Figure 6). The con-
centrations N
1
and N
2
varied from sample to sample,
reflecting that total VLP production was dependent on
sample-dependent factors, such as the initial cell density.
However, the population fraction f
2
= N
2
/(N
1
+N
2

)
remained approximately constant for all measured sam-
ples, f
2
=19±7%.Thus,apopulationof~19%ofthe
VLPs is associated with the higher Gag copy number.
Note that a similar heterogeneity in Gag copy numbers
has also been reported for HIV-1 VLPs [21].
Discussion
Recent progress in cryo-TEM, cryo-ET and 3 D recon-
struction has led to many major breakthroughs in our
understanding of virus structure. For instance, the archi-
tecture of immature and matur e HIV-1 [13,23,24], mur-
ine leukemia virus (MuLV) [25], and Rous sarcoma
virus (RSV) [26] has been investigated in great detail.
Although HTLV-1 was the first human retrovirus to be
discovered [27,28], v ery little is known about HTLV-1
virion morphology . Progress in this are a of HTLV-1
Radius (Å)
n=14
Density
-30
-20
-10
0
10
20
30
40
100 200 300 400 500 600

Density
STD
Figure 4 Radial density profile of the HTVL-1-like particles. The solid line represents the average density measured; dashed line indicates the
standard deviation (n = 14).
Grigsby et al. Retrovirology 2010, 7:75
/>Page 7 of 13
biology has been hampered due to the fragile nature of
HTLV-1 proviral sequences as well as limited levels of
viral gene expression and viral replication in tissue cul-
ture. HTLV-1 pathogenesis is typically observed decades
after infection with low viral loads. In fact, studies have
shown that HTLV-1 restricts its own gene expression
via viral regulatory factors [29,30]. HTLV-1 has likely
evolved such a replication strategy for immune escape.
Furthermore, high AU-content of the retroviral genome
may lead to instability during nuclear transport of
mRNAs [31], which also contributes to the overall low
level of viral gene and protein expression. In this study,
we have designed a model system to study HTLV-1 Gag
trafficking in cells and VLP production and morphology.
The basis for this model system is a codon-optimized
HTLV-1 Gag-EYFP construct, which can be readily
amplified as a plasmid, express es high levels of HTLV-1
Gag in mammalian cells, and robustly produces VLPs.
This is the first model system developed for HTLV-1
for the study of virus particle assembly, release, as well
as virus particle morphology.
While our model system does not express Gag in the
context of a proviral sequence (i.e., codon-optimized
and EYFP-tagged), our results indicate that the VLPs

produced have the morphology of the authentic HTLV-
1 immature particles . Furthermore, while the Gag traf-
ficking pathways used by the HTLV-1 Gag in this model
system may be different from that of Gag expressed
from the provirus, the production of VLPs argues that
the trafficking pathways are biologica l relevant since
VLP production is the result of expression of the
codon-optimized Gag-EYFP fusion. The altered Gag
A
Intensity (ms
-1
)
50
100
150
200
250
Time (s)
0
200
400
600 800 1000
B
Photon counts (k)
5
10 15
20
0
p(k)
10

0
10
-2
10
-4
10
-6
10
-8
Res
2
0
-2
-4
C
G( )
(ms)
0
1
2
3
4
-1
0.01
0.10
1
10 100
100001000
Figure 5 Fluorescence fluctuation spectroscopy analysis of
HTLV-1 Gag-based VLPs. (A). The fluorescence intensity trace

shows discrete peaks, which correspond to individual VLPs diffusing
through the observation volume. (B). Experimental photon counting
histogram (diamonds) of the VLP sample. A fit (solid line) of the
histogram to a 2-species model with background identifies the
concentration and Gag copy number of the VLPs. The presence of
two species indicates the existence of heterogeneity in the Gag
copy number of VLPs. A weighted average of the two species leads
to an average Gag copy number of 530 per VLP. The first VLP
species has a copy number of 270 and a concentration of 20.5 pM.
The second VLP species, which is brighter than the first, has a copy
number of 800 and a concentration of 6.5 pM. (C). A fit (solid line)
of the autocorrelation function (diamonds) to a diffusion model
identifies an average hydrodynamic diameter of 74 nm for the VLPs.
A
Mean
Sub-population 1
Sub-population 2
Gag copy number
200
400
600
800
1000
1200
0
Figure 6 Gag copy number of HTLV-1 Gag based VLPs. The Gag
copy number was determined by FFS analysis of several
independent VLP samples (n = 5). The mean copy number per VLP
is shown together with the corresponding copy number of the two
subpopulations identified by FFS analysis. The error bars represent

the standard deviation of the multiple measurements.
Grigsby et al. Retrovirology 2010, 7:75
/>Page 8 of 13
trafficking pathways could influence envelope incorpora-
tion into VLPs, though our cryo-TEM data revealed an
abundance of VLPs with VSVG. The VLPs characterized
in our study resemble immature HTLV-1 and can be
readily observed in ultrathin sections of 293Ts trans-
fected with pEYFP-N3 HTLV-1 Gag (Figure 1C). In
addition, cell culture supernatants from 293Ts transi-
ently transfected wit h pEYFP-N3 HTLV-1 Gag contain
high levels of Gag-EYFP fusion proteins (Figure 1B),
which provides second line of evidence for the produc-
tion of VLPs. In the fraction of sucrose gradients con-
taining the highly-fluorescent materia l, cryo-TEM
reveals that these fractions are highly concentrated with
VLPs (Figure 3A). Expression of EYFP alone in cells did
not lead to the release of fluorescence in the cell culture
supernatant (data not shown), arguing that we were spe-
cifically detecting the Gag-EYFP fusion in the VLPs.
We found that the intracellular localization of HTLV-1
Gag-EYFP was comparable to that of authentic Gag in
HeLa cells (Figure 2). This implies, though does not for-
mally prove, that there are similarities in the Gag traffick-
ing pathway used by Gag-EYFP and authentic Gag.
Among retroviruses, intracellular Gag polyproteins are
thought to target and accumulate at membrane compart-
ments prior to viral assembly. In the case of HIV-1, Gag is
thought to primarily target specific domains of the plasma
membrane where PI(4,5)P2 and cholesterol are enriched,

though endosomal trafficking may also play a role. For
HTLV-1, several studies have suggested the association of
Gag with several markers found on the membranes of late
endosomes and multivesicular bodies - these markers are
also enriched at the plasma membrane [32-35].
Our cryo-TEM and FFS analysis determined that the
average VLP diameter was 71 ± 20 nm and 75 ± 4 nm,
respectively. As observed in other retroviruses, the size
of HTLV-1-like particles varies greatly, ranging from 30
to 237 nm. According to the size distribution (Figure
3B), over 25% of the population is between 70-80 nm in
diameter, indicating that H TLV-1 is smaller, on average,
than previously believed. The average diameter of
HTLV-1 has been shown to be anywhere from 95.1 ±
19.0 nm to 110.0 ± 15.5 nm depending on different
types of staining used for TEM [19]. However, morpho-
logical details are lost with staining methods when the
biological specimens are completely dehydrated. Exam-
ining frozen, hydrated samples via cryo-TEM reflects
the native morphology of the viral particles. Moreover,
FFS offers a unique way to determine the average hydro-
dynamic radius in the cell supernatant without any spe-
cial treatment or preparation prior to FFS analysis. The
use of t wo independent methods fo r determining VLP
diameter provides a strong argument in favor of the
relatively small particle diameter for the HTLV-1-like
particles analyzed in our study.
We used FFS to also investigate Gag stoichiometry in
the VLPs by performing brightness analysis of the FFS
data. We determined that the average Gag copy number

per VLP is ~510, which implies that only half of the
available membrane surface is covered by Gag. Bright-
ness analysis furthe r revealed heterogeneity of the Gag
copy number by identifying two brightness species. The
presence of heterogeneityintheGagcopynumberhas
also been observed for HIV-1 Gag-based VLPs [21].
Since FFS analysis involves an ensemble average over all
measured VLPs, the information in the PCH curve only
provides a rough approximation of the true Gag copy
number distribution for th e VLPs. Thus, the two bright-
ness species identified by PCH analysis do not necessa-
rily reflect two distinct populations of VLPs, but more
likely reflect the analytical approximation of a broad dis-
tribution of Gag stoichiometries that appro ximately
range from 300 to 880. PCH analysis also demonstrates
that only ~20% of VLPs have high copy numbers.
Among the thousands of cryo-TEM images of VLPs
examined in our study, we commonly observed particles
that did not have electron density consistent with a Gag
shell covering the entire membrane surface (Figure 3A).
These results suggest that the majority of HTLV-1 parti-
cles analyzed contain an i ncomplete shell of G ag lattice.
In the case of HIV-1, previous 3 D structural analyses
revealed that most immature virions contain a continu-
ous, but incomplete, hexameric arranged Gag shell, cov-
ering approximately 40-60% of the membrane surface
[14,15,36]. The average copy number of Gag per particle
was calculated to be approximately 2,400 ± 700 per
immature particle. The Gag number increased signifi-
cantly, however, w hen defects were introduced during

budding [ 36]. In fact, the data is in agreement with our
previous FFS study indicating that HIV-1 Gag stoichio-
metry ranges from 750 to 2,500 [21]. Since the mature
core consists of only 1,000-1,500 molecules of CA [23],
it is reasonable to believe that an equivalent number of
Gag molecules are needed to form an immature particle.
Our current study is the first to provide insights into the
structural details for HTLV-1.
In vitro studies suggest that the HTLV-1 Gag shell is
very likely to be organized differently compared to that
of HIV-1 Gag [16-18]. When examining the cryo-TEM
images of HTLV-1-like particles, we rarely observed a
hig hly ordered Gag lattice next to the lipid bilayer (Fig-
ure 3A), a feature frequently observed in immature
HIV-1 particles. T he HTLV-1 particles analy zed in our
study were fairly uniform in their overall inner density.
Furthermore, in contrast to HIV-1, no defined peaks
representing the CA or NC domains were found in the
HTLV-1 radial density profile. The two peaks represent-
ing the lipid bilayers could be clearly determined (Figure
4), whereas the in ner density profile appeared to be
Grigsby et al. Retrovirology 2010, 7:75
/>Page 9 of 13
relatively flat. Since cryo-TEM images represent a two-
dimensional projectio n of the virus particle, a more rig-
orous structural analysis, such as cryo-ET, is needed to
further examine the protein organization in the HTLV-
1-like particles.
In summary, we have developed the first efficient a nd
robust model system for the analysis of HTLV-1 Gag

cellular trafficking, virus particle assembly, release and
particle morphology. This system will allow for signifi-
cant advancements in understanding of the basic
mechanisms of HTLV-1 replication - which has been
severely hampered due to the limitations in studying
HTLV-1 in tissue culture. Our study also represents the
first description of immatur e HTLV-1 particles as well
as quantitative measurements of particle size, Gag copy
number, and an initial analysis of the HTLV-1 Gag lat-
tice. Future application of cryo-electron tomography will
aid in gaining greater insight into HTLV-1 particle mor-
phology. A deeper understanding of the basic mechan-
isms involved in HTLV-1 particle assembly and
morphology should help to enhance our global under-
standing of the basis of HTLV-1 particle infectivity,
transmission and pathogenesis.
Materials and methods
Construction of codon-optimized HTLV-1 gag-yfp fusion
A codon-optimized HTLV-1 Gag gene was designed
using the UpGene program [37] and synthesized by Gen-
Script Co. (Piscataway, NJ). The synthetic HTLV-1 gag
contains an optimal Kozak consensus sequence [38,39] at
the 5′ end of the gene: GCCACCATGG (start codon in
bold). Two restriction enzyme sites, Hind III and Bam
HI, were also engineered into the 5′ and 3′ end of the
gene, respectively, for sub-cloning purposes. For reporter
gene construction, the artificial HTLV-1 gag was cloned
into a pEYFP-N3 vector using the HindIII and BamHI
restriction sites, creating pEYFP-N3 HTLV-1 Gag.
Immunoblotting

293T cells were transiently transfected with the pEYFP-N3
HTLV-1 Gag construct using GenJet (SignaGen, Gaithers-
burg, MD) according to the manufacturer’sinstructions.
Thirty-six hours post-transfection, cell pellets and super-
natant were collected and lysates were prepared as pre-
viously described [40]. Lysates were subjected to
electrophoresis on 12.5% polyacrylamide gels and trans-
ferred to nitrocellulose (Bio-Rad, Hercules, CA). HTLV-1
Gag polyprotein was detected with a primary mouse anti-
HTLV-1 p24 antiserum (Abcam, Cambridge, MA) at
1:1500 dilution followed by a horseradish peroxidase-con-
jugated goat anti-mouse IgG (Thermo Fisher, Rockford,
IL) at 1:5000 dilution. Gag polyprotein expression was
detected with a ChemiDoc XRS system (Bio-Rad).
Immunofluorescence and fluorescence microscopy
HeLa cells were grown on Lab-Tek II chamber slides
(Fisher Scientific) and transfected with either the
pEYFP-N3 HTLV-1 Gag construct or a HTLV-1 proviral
clone (a kind gift from Dr. Marie-Christine Dokhelar)
[41]. Thirty-six hours post-transfect ion, cells were
washed twice with 1× PBS buffer and fixed with 4% par-
aformaldehyde for 20 min. For cells transfected with
pEYF P-N3 HTLV-1, cells were washed three times after
fixation, and stained for 5 min with 1 μg/ml DAPI
(Sigma-Aldrich, St. Louis, MO) in 1× PBS containing
0.05% Triton X-100 (Sigma-Aldrich), then preserved
using ProLong Gold antifade mount ing regent (Invitro-
gen, Carlsbad, CA). For cells transfected with the
HTLV-1 proviral clone, permea bilization was achieved
by treating with 1× PBS containing 0.5% Triton X-100

for 2 min at room temperature following fixation. Cells
were then washed three times and blocked with 1× PBS
containing 5% normal donkey serum (Sigma-Aldrich)
for 30 min. Primary mouse anti-HTLV-1 p24 antisera
(Abcam) were diluted (1:150) in blocking solution and
incubated with cells. After incubation for 2 hr at room
temperature, cells were washed three times, followed by
a second incubation for 1 hr at room temperature with
diluted (1:250) Alexa Fluor 488-conjugated donkey anti-
mouse IgG (Invitrogen). Prior to mounting, cells were
washed five times and stained with DAPI as described
above. Intracellular local ization of Gag polyprotein was
detected with an Oly mpus FV500 confocal laser scan-
ning microscope. Optical sections of cells were collected
with a Plan-Apo 60×/1.45 NA TIRFM objective at 1.5
zoom. The z- series were reconstructed using Olympus
FluoView software.
VLPs purification for cryo-TEM
293T cells were co-transfected with pEYFP-N3 HTLV-1
and a vesicular stomatitis virus G (VSV-G)protein
(10:1) expression construct using Ge neJet. Twenty-four
hours post-transfection, the cell culture media was
changed to a serum-free media and incubated for an
additional 12 hr. In order to harvest VLPs, tissue culture
supernatant was centrifuged at 3000 × g for 5 min to
remove large cellular debris, then the supernatant was
passed through an Amicon Ultra- 15 Centrifugal Filter
Unit (100 KDa) (Millipore, Bil lerica, MA) to concentrate
samples. The concentrated samples were then subje cted
to a 10-40% linear sucrose gradient prepared with a

Gradient Master (BioComp, Fredericton, NB, Canada).
Samples were then ultracentrifuged at 35,000 rpm for
30 min at 4°C using a SW55 Ti rotor. The VLP fraction
was extracted and pelleted at 35,000 rpm, 4°C for 1.5 hr
using a SW55 Ti rotor (Beckman). After centrifugation,
the pellet was resuspended in 1× STE buffer (10 mM
Grigsby et al. Retrovirology 2010, 7:75
/>Page 10 of 13
Tris-Cl, pH 7.4, 100 mM NaCl, 1 mM EDTA) at 4°C for
4 hr and then analyzed by cryo-TEM.
TEM of transfected cells
293T cells were transfected with either pEYFP-N3
HTLV-1 or a HTLV-1 proviral clone as described
above. Thirty-six hours post-transfection, cells were har-
vested and washed twice with 1× PBS followed by an
addition al wash in 0.1 M sodium cacodylate. To prepare
thin sections, cell pellets were first fixed with 2.5% glu-
taraldehyde for 40 min and then washed three times
with 0.1 M sodium cacodylate. After washing, the sam-
ples were post-fixed with 1% OsO
4
for 30 min, followed
by three rinses. The samples were then subjected to
increasing concentrations of ethanol for dehydration.
Immediately following the application of 70% ethanol,
en bloc staining was added to the samples for 30 min
before embedding in Epon 812 resin. Ult rathin sections
(65 nm) were acquired and stained with uranyl acetate
and lead citrate, then examined by electron microscopy
using a JEOL 1200EX transmission electron microscope.

Cryo-TEM of HTLV-1 VLPs and calculation of radial profile
A 3ul aliquot of the purified and concentrated HTLV-1
VLP sample preparatio n was applied to a glow-dis-
charged c-flat holey carbon grid (Ted Pella, Redding,
CA) and used for plunge freezing into liquid ethane [42]
with a FEI Vitrobot MarkIII system. The frozen grids
were then transferred to a FEI TF30 field emission gun
transmission electron microscope at liquid nitrogen
temperature. Images were recorded at a magnification of
40kto100katlow-dose(~30e/Å2)and1to5μm
underfocus conditions using a Gatan 4 k by 4 k CCD
camera.
In order to calculate the radial density profile, images
of VLPs with spherical morphology were boxed using
RobEM />bem.txt. The center of each particle was determined
using the p rogram EMCORORG that calculates cross-
correlation of each image with its 180° rotational image
The radial profile
for each particle was then calculated by computing the
rotationally averaged density relative to the center of the
particle. A group of 14 VLP images with a diameter in
the range of 70-80 nm was used for calculation of the
averaged radial profile. Each pixel in the image c orre-
sponds to a 3.0-Å spacing in the VLP. The defocus
levels of these images were between 1.5-3.7 μm, which
allows for visualization of both membrane leaflets of the
viral membrane. The radial profile of each particle was
first calculated to obtain the highest density position of
the outer membrane leaflet. The radial p rofile of each
particle was then linearly interpreted to match the posi-

tion of the outer membrane to the averaged position
(367-Å radius). The average radial profile and standard
deviation were then calculated.
VLP size measurements
Cryo-TEM i mages were analyzed using ImageJ software
(NIH, Bethesda, MD). For each VLP, two perpendicular
diameters were used to calculate the average diameter.
The histogram was generated using GraphPad Prism 5
software (GraphPad, La Jolla, CA).
VLP preparation and FFS experimental setup
293T cells were co-transfected with pEYFP-N3 HTLV-1
and a VSV-G expression construct (10:1) as described
earlier. Aliquots of the cell culture supernatants used for
subsequent cryo-TEM analysis were removed for parallel
analysis by FFS. Thirty-six hours post-transfection, VLPs
were harvested and clarified of cellular debris by low-
speed centrifugation at 3000 × g for 5 min as well as
passing through a 0.22 μm filter. The resulting clarified
supernatants were then used for FFS measurements.
A mode-locked Ti:sapphire laser (Tsunami, Spectra-
Physics, Mountain View, CA) pumped by an intracavity
doubled Nd:YVO4 laser (Millenia, Spectra Physics) is
the source of two-photon excitation. Experi ments were
performed on a modified Zeiss Axiovert 200 microscope
(Thornwood, NY) as previously described [22]. Each FFS
measurement collects data at a sampling frequency of
20 kHz for a duration of 20-30 min at an excitation
wavelength of 905 nm. The viral particles are measured
using a 63× C-Apochromat water immersion objective
(N.A. = 1.2). The excitation power at the objective ran-

ged from 0.1-0.4 mW. A volume of 200 μ l of VLP solu-
tion was added to an 8-well Nunc Lab-Tek Chamber
Slide mounted on the microscope. To avoid evaporation
unused wells were filled with water and the slide was
closed with a lid. Measurements were taken 10 μm
above the bottom of the well.
FFS data analysis
A brief description of the analysis method is provided
here. A d etailed discussion of FFS analysis of VLP sam-
ples can be found elsewhere [21]. The diffusion time
was deter mined by fitting the calculated autocorrelation
function to a single species diffusion model [43]. The
ratio of diffusion time for the two samples is equated,
according to the Stokes-Einstein relati on, to the ratio of
the hydrodynamic radii of the diffusi ng particles τ
D1
/τ
D2
=r
1
/r
2
. The measured diffusion t ime of fluorescent
sphere s with a known radius of 50 nm serves as a refer-
ence to calculate the average diameter of the VLPs [44].
The FFS data was also fit to a 3-species PCH model
with deadtime and afterpulsing corrections [45]. Each
independent species in PCH is defined by its brightne ss
ε and the av erage number N of particles in the optical
Grigsby et al. Retrovirology 2010, 7:75

/>Page 11 of 13
observation volume. The particle number N is converted
into a concentration after the observation volume is
calibrated with a dye sample. One of the three sp ecies is
required to take the aut o-fluorescent background of the
solution into account as discussed in a recent paper on
HIV-1 VLPs [21]. This background species, which has a
vanishingly small brightness, is included in every PCH
fit and will not be reported. The other two species of
the PCH model define the VLP sample. The presence of
two brightness species indicates the existence of Gag
copy number heterogeneity within the HTLV-1 VLP
population as previously observed for HIV-1 VLPs [21].
PCH fitting was carried out by programs written in IDL
6.4 (Research Systems, Boulder CO). Error analysis of
FFS data was carried out as previously described [46].
FFS brightness calibration and experimental
considerations
To avoid unwanted optical effects, all experiments are
conducted in a power range where the fluorescence inten-
sity of YFP scales quadratically with excitation power. We
also confirmed that within this power range the average
occupation number N remains constant, which establishes
a constant optical observation volume. The brightness of a
protei n complex scales with the number of YFP-labels it
contains [21]. The YFP copy number of a complex is
determined by the normalized brightness b = ε/ε
YFP
,
where ε

YFP
is the brightness of the YFP monomer and ε is
the brightness of the complex. A calibration measurement
of YFP brightness, ε
YFP
, is necessary to determine copy
number. Because YFP brightness is difficult to determine
at the low powers that the VLPs must be measured at to
avoid saturation of the detector, the YFP brightness, ε
high
,
is measured at a higher excitation power, P
high
. Conversion
to the YFP brightness ε
low
for the low power P
low
of VLP
experiments is achieved by using the relationship of power
to brightness ε
low
= ε
high
(P
low
2
/P
high
2

). The data acquisition
time for the VLP measurements was chosen such that at
least 1000 VLP s passed through the observation volume,
which is sufficient for statistical analysis of the data. All
VLP measurements were performed at excitation powers
that are free from sat uration an d bleaching art ifacts [21].
The FFS experiments identified two brightness species for
the VLP sample. The average normalized brightness b
avg
of the two species is determined by a non-linear relation-
ship [22],
b
bN bN
bN b N
avg
,=
+
+
1
2
1
2
2
2
11 2 2
where b
i
and N
i
are the normalized brightness and the

number of particles in the observation volume of each
species.
Additional material
Additional file 1: Supplemental Figure 1. Low magnification cryo-TEM
image of VLPs produced from 293T cells. Image provides another
example of the types of particles observed by cryo TEM. Scale bar = 100
nm.
Acknowledgements
We are grateful to Ms. Fang Zhou for the assistance of TEM. TEM and cryo-
TEM were conducted at the University of Minnesota Characterization Facility.
The computational studies of HTLV-1 cryo-TEM images were done at the
Minnesota Supercomputing Institute. We thank Dr. Paul Jardine for technical
advice and Dr. Christine Clouser for critical reading of the manuscript. I. F. G.
was supported by NIH T32 DE07288 (MinnCResT Program), and K. F. by T32
CA09138 (Cancer Biology Training Grant). This work is supported by NIH
Grants R01GM064589 (J.D.M) and R21AI81673 (J.D.M., Y.C. and L.M.M.).
Author details
1
Institute for Molecular Virology, University of Minnesota, Minneapolis, MN
55455, USA.
2
Department of Diagnostic and Biological Sciences, School of
Dentistry, University of Minnesota, Minneapolis, MN 55455, USA.
3
Department of Microbiology, Medical School, University of Minnesota,
Minneapolis, MN 55455, USA.
4
School of Physics and Astronomy, University
of Minnesota, Minneapolis, MN 55455, USA.
Authors’ contributions

IFG, WZ, JLJ, KF, YC and JR carried out the experimental work, participated in
the data analysis and interpretation, and contributed in the writing of the
manuscript. WZ, JDM, YC and LMM conceived of the study, oversaw
experimental design, data analysis, and interpretation as well as edited the
manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 2 July 2010 Accepted: 20 September 2010
Published: 20 September 2010
References
1. Proietti FA, Carneiro-Proietti AB, Catalan-Soares BC, Murphy EL: Global
epidemiology of HTLV-I infection and associated diseases. Oncogene
2005, 24:6058-6068.
2. Gessain A, Barin F, Vernant JC, Gout O, Maurs L, Calender A, de The G:
Antibodies to human T-lymphotropic virus type-I in patients with
tropical spastic paraparesis. Lancet 1985, 2:407-410.
3. Osame M, Usuku K, Izumo S, Ijichi N, Amitani H, Igata A, Matsumoto M,
Tara M: HTLV-I associated myelopathy, a new clinical entity. Lancet 1986,
1:1031-1032.
4. Gheysen D, Jacobs E, de Foresta F, Thiriart C, Francotte M, Thines D, De
Wilde M: Assembly and release of HIV-1 precursor Pr55gag virus-like
particles from recombinant baculovirus-infected insect cells. Cell 1989,
59:103-112.
5. Briggs JA, Simon MN, Gross I, Krausslich HG, Fuller SD, Vogt VM,
Johnson MC: The stoichiometry of Gag protein in HIV-1. Nat Struct Mol
Biol 2004, 11:672-675.
6. Lanman J, Lam TT, Emmett MR, Marshall AG, Sakalian M, Prevelige PE Jr:
Key interactions in HIV-1 maturation identified by hydrogen-deuterium
exchange. Nat Struct Mol Biol 2004, 11:676-677.
7. Benjamin J, Ganser-Pornillos BK, Tivol WF, Sundquist WI, Jensen GJ: Three-

dimensional structure of HIV-1 virus-like particles by electron
cryotomography. J Mol Biol 2005, 346:577-588.
8. Ono A, Orenstein JM, Freed EO: Role of the Gag matrix domain in
targeting human immunodeficiency virus type 1 assembly. J Virol 2000,
74:2855-2866.
9. Ono A, Ablan SD, Lockett SJ, Nagashima K, Freed EO: Phosphatidylinositol
(4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma
membrane. Proc Natl Acad Sci USA 2004, 101:14889-14894.
Grigsby et al. Retrovirology 2010, 7:75
/>Page 12 of 13
10. Bryant M, Ratner L: Myristoylation-dependent replication and assembly of
human immunodeficiency virus 1. Proc Natl Acad Sci USA 1990,
87:523-527.
11. Riffel N, Harlos K, Iourin O, Rao Z, Kingsman A, Stuart D, Fry E: Atomic
resolution structure of Moloney murine leukemia virus matrix protein
and its relationship to other retroviral matrix proteins. Structure 2002,
10:1627-1636.
12. Usami Y, Popov S, Popova E, Inoue M, Weissenhorn W, H GG: The ESCRT
pathway and HIV-1 budding. Biochem Soc Trans 2009, 37:181-184.
13. Fuller SD, Wilk T, Gowen BE, Krausslich HG, Vogt VM: Cryo-electron
microscopy reveals ordered domains in the immature HIV-1 particle.
Curr Biol 1997, 7:729-738.
14. Briggs JA, Riches JD, Glass B, Bartonova V, Zanetti G, Krausslich HG:
Structure and assembly of immature HIV. Proc Natl Acad Sci USA 2009,
106:11090-11095.
15. Wright ER, Schooler JB, Ding HJ, Kieffer C, Fillmore C, Sundquist WI,
Jensen GJ: Electron cryotomography of immature HIV-1 virions reveals
the structure of the CA and SP1 Gag shells. Embo J 2007, 26:2218-2226.
16. Bertola F, Manigand C, Picard P, Belghazi M, Precigoux G: Human T-
lymphotrophic virus type I nucleocapsid protein NCp15: structural study

and stability of the N-terminal zinc-finger. Biochem J 2000, 352(Pt
2):293-300.
17. Christensen AM, Massiah MA, Turner BG, Sundquist WI, Summers MF:
Three-dimensional structure of the HTLV-II matrix protein and
comparative analysis of matrix proteins from the different classes of
pathogenic human retroviruses. J Mol Biol 1996, 264:1117-1131.
18. Cornilescu CC, Bouamr F, Yao X, Carter C, Tjandra N: Structural analysis of
the N-terminal domain of the human T-cell leukemia virus capsid
protein. J Mol Biol 2001, 306:783-797.
19. Doultree JC, Kiernan RE, Lee JY, Bowden DS, McPhee DA, Tokuyasu KT,
Marshall JA: A new electron microscope positive staining method for
viruses in suspension. J Virol Methods 1992, 37:321-335.
20. Wills JW, Craven RC: Form, function, and use of retroviral gag proteins.
AIDS 1991, 5:639-654.
21. Chen Y, Wu B, Musier-Forsyth K, Mansky LM, Mueller JD: Fluorescence
fluctuation spectroscopy on viral-like particles reveals variable gag
stoichiometry. Biophys J 2009, 96:1961-1969.
22. Chen Y, Wei LN, Muller JD: Probing protein oligomerization in living cells
with fluorescence fluctuation spectroscopy. Proc Natl Acad Sci USA 2003,
100:15492-15497.
23. Briggs JA, Wilk T, Welker R, Krausslich HG, Fuller SD: Structural organization
of authentic, mature HIV-1 virions and cores. Embo J 2003, 22:1707-1715.
24. Briggs JA, Grunewald K, Glass B, Forster F, Krausslich HG, Fuller SD: The
mechanism of HIV-1 core assembly: insights from three-dimensional
reconstructions of authentic virions. Structure 2006, 14:15-20.
25. Nermut MV, Mulloy B: Consideration of the three-dimensional structure
of core shells (capsids) in spherical retroviruses. Micron 2007, 38:462-470.
26. Briggs JA, Johnson MC, Simon MN, Fuller SD, Vogt VM: Cryo-electron
microscopy reveals conserved and divergent features of gag packing in
immature particles of Rous sarcoma virus and human immunodeficiency

virus. J Mol Biol 2006, 355:157-168.
27. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC: Detection
and isolation of type C retrovirus particles from fresh and cultured
lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl
Acad Sci USA 1980, 77:7415-7419.
28. Poiesz BJ, Ruscetti FW, Reitz MS, Kalyanaraman VS, Gallo RC: Isolation of a
new type C retrovirus (HTLV) in primary uncultured cells of a patient
with Sezary T-cell leukaemia. Nature 1981, 294:268-271.
29. Gaudray G, Gachon F, Basbous J, Biard-Piechaczyk M, Devaux C,
Mesnard JM: The complementary strand of the human T-cell leukemia
virus type 1 RNA genome encodes a bZIP transcription factor that
down-regulates viral transcription. J Virol 2002, 76:12813-12822.
30. Clerc I, Polakowski N, Andre-Arpin C, Cook P, Barbeau B, Mesnard JM,
Lemasson I: An interaction between the human T cell leukemia virus
type 1 basic leucine zipper factor (HBZ) and the KIX domain of p300/
CBP contributes to the down-regulation of tax-dependent viral
transcription by HBZ. J Biol Chem 2008, 283:23903-23913.
31. Schwartz S, Felber BK, Pavlakis GN: Distinct RNA sequences in the gag
region of human immunodeficiency virus type 1 decrease RNA stability
and inhibit expression in the absence of Rev protein. J Virol 1992,
66:150-159.
32. Blot V, Perugi F, Gay B, Prevost MC, Briant L, Tangy F, Abriel H, Staub O,
Dokhelar MC, Pique C: Nedd4.1-mediated ubiquitination and subsequent
recruitment of Tsg101 ensure HTLV-1 Gag trafficking towards the
multivesicular body pathway prior to virus budding. J Cell Sci 2004,
117:2357-2367.
33. Dorweiler IJ, Ruone SJ, Wang H, Burry RW, Mansky LM: Role of the human
T-cell leukemia virus type 1 PTAP motif in Gag targeting and particle
release. J Virol 2006, 80:3634-3643.
34. Mazurov D, Heidecker G, Derse D: HTLV-1 Gag protein associates with

CD82 tetraspanin microdomains at the plasma membrane. Virology 2006,
346:194-204.
35. Mazurov D, Heidecker G, Derse D: The inner loop of tetraspanins CD82
and CD81 mediates interactions with human T cell lymphotrophic virus
type 1 Gag protein. J Biol Chem 2007, 282:3896-3903.
36. Carlson LA, Briggs JA, Glass B, Riches JD, Simon MN, Johnson MC, Muller B,
Grunewald K, Krausslich HG: Three-dimensional analysis of budding sites
and released virus suggests a revised model for HIV-1 morphogenesis.
Cell Host Microbe 2008, 4:592-599.
37. Gao W, Rzewski A, Sun H, Robbins PD, Gambotto A:
UpGene: Application
of a web-based DNA codon optimization algorithm. Biotechnol Prog 2004,
20:443-448.
38. Kozak M: Compilation and analysis of sequences upstream from the
translational start site in eukaryotic mRNAs. Nucleic Acids Res 1984,
12:857-872.
39. Kozak M: An analysis of 5′-noncoding sequences from 699 vertebrate
messenger RNAs. Nucleic Acids Res 1987, 15:8125-8148.
40. Wang H, Norris KM, Mansky LM: Involvement of the matrix and
nucleocapsid domains of the bovine leukemia virus Gag polyprotein
precursor in viral RNA packaging. J Virol 2003, 77:9431-9438.
41. Le Blanc I, Rosenberg AR, Dokhelar MC: Multiple functions for the basic
amino acids of the human T-cell leukemia virus type 1 matrix protein in
viral transmission. J Virol 1999, 73:1860-1867.
42. Baker TS, Olson NH, Fuller SD: Adding the third dimension to virus life
cycles: three-dimensional reconstruction of icosahedral viruses from
cryo-electron micrographs. Microbiol Mol Biol Rev 1999, 63:862-922, table
of contents.
43. Thompson NL, Lieto AM, Allen NW: Recent advances in fluorescence
correlation spectroscopy. Current Opinion in Structural Biology 2002,

12:634-641.
44. Wu B, Chen Y, Muller JD: Fluorescence correlation spectroscopy of finite-
sized particles. Biophys J 2008, 94:2800-2808.
45. Hillesheim LN, Müller JD: The Photon Counting Histogram in
Fluorescence Fluctuation Spectroscopy with Non-Ideal Photodetectors.
2003, 85:1948-1958.
46. Müller JD, Chen Y, Gratton E: Resolving Heterogeneity on the Single
Molecular Level with the Photon-Counting Histogram. 2000, 78:474-486.
doi:10.1186/1742-4690-7-75
Cite this article as: Grigsby et al.: Biophysical analysis of HTLV-1 particles
reveals novel insights into particle morphology and Gag stoichiometry.
Retrovirology 2010 7:75.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Grigsby et al. Retrovirology 2010, 7:75
/>Page 13 of 13